5.2 M ETHODEN
5.2.6 Kultur von Insektenzellen
5.2.6.3 Transfektion rekombinanter viraler DNA in Insektenzellen
Für die Transfektion mit rekombinanter Bacmid-DNA wurden adhärent wachsende SF9-Zellen, die zu 80% konfluent gewachsen waren, verwendet. Diese Zellen wurden einmal mit Transfektionsmedium gewaschen und mit 5 ml Transfektionsmedium überschichtet, in das
werden vermischt mit 50 µ Lipofektin in 500 µl Transfektionsmedium und 30 Minuten bei RT inkubiert) gegeben wurde. Nach fünf Stunden Inkubation bei 27 °C wurde das Transfektionsmedium durch 10 ml Vollmedium ersetzt. Es folgte eine Inkubation von für 72 h bei 27 °C, nach der das virenhaltige Medium (=Virenstocklösung) abgenommen wurde. Die Lagerung der Virenstocklösung erfolgte bei -70 °C. Um einen ausreichenden Virentiter für die Proteinexpression zu erhalten, musste die Virenstocklösung amplifiziert werden: Hierzu wurde ein Aliquot der Virenstocklösung auf 80% konfluente Zellen gegeben, die dann für 72 h bei 27 °C inkubiert wurden. Anschließend wurde das virenhaltige Medium abgenommen und bei 4°C oder –20 °C gelagert.
Die Proteinexpression sollte in Spinnerflaschen in Suspensionskulturen mit einem Volumen von 50 ml ablaufen. Leider starben die SF9-Zellen schon nach Transfektion mit dem kringelchen-enthaltenden Bacmid ab, so dass keine Proteinexpression erfolgen konnte.
6 Abkürzungen
A Adenin
Amp Ampicillin
AP alkalische Phosphatase
bp Basenpaare
BCIP 5-Brom-4-chlor-3-indolylphosphat BLAST „Basic Local Alignment Search Tool”
BSA Rinderserumalbumin
C Cytosin
cDNA komplementäre DNA
CHAPS 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propansulfonat
Da Dalton
DAG Diacylglycerol
dATP Desoxyadenosinnukleotidtriphosphat dCTP Desoxycytosinnukleotidtriphosphat DEPC Diethylpyrocarbonat
ddH2O doppeldestiliertes Wasser
DIG Digoxygenin
DMSO Dimethylsulfoxid
DNA Desoxyribonukleinsäure DNase Desoxyribonuklease dNTP Desoxynukleotidtriphosphat ddNTP Didesoxynukleotidtriphosphat DTT Dithiothreitol
EDTA Ethylendiamintetraessigsäure EST(s) “Expressed Sequence Tag(s)“
EtBr Ethidiumbromid
EtOH Ethanol
FGF Fibroblasten-Wachstumsfaktor
FGFR FGF-Rezeptor
FKS fötales Kälberserum
G Guanin
g Gramm
hFGFR humaner FGF-Rezeptor
HCl Salzsäure
IPTG Isopropylthiogalaktosid I-Zellen interstitielle Zellen
kb Kilobasenpaare
kV Kilovolt
LBAMP LB-Medium mit 100 µg/ml Ampicillin LiCl Lithium-Chlorid
M molar
mA Milliampère
MAB Maleinsäurepuffer MAB-B Maleinsäurepuffer mit 1% BSA
max. maximal
MCS “multiple cloning site” oder Polylinker
MeOH Methanol
µg Mikrogramm
µl Mikroliter
µM mikromolar
mg Milligramm
ml Milliliter
mM millimolar
mol Mol
mRNA Messenger-RNA
NaOH Natronlauge
NBT Nitrotetrazoliumblauchlorid
NCBI „National Center for Biotechnology Information“
Ni-Agarose Nickel Nitrilotriacetic Acid Agarose
OD600 Optische Dichte bei 600 nm
Ω Ohm
PAA Polyacrylamid
PBS Phosphat-gepufferte Salzlösung PCR Polymerase-Kettenreaktion PEG Polyethylenglykol
RNA Ribonukleinsäure
RNase Ribonuklease
rpm „rounds per minute“
rRNA ribosomale RNA
RT Raum-Temperatur oder Reverse Transkription RT-PCR Reverse Transkriptions-PCR
SDS Natriumdodecylsulfat
ss “single stranded” (einzelsträngige)
T Thymin, Temperatur
Tab. Tabelle
Taq Thermophilus aquaticus TBE Tris-Borat-EDTA-Puffer TCA Trichloressigsäure
TEMED N,N,N´,N´- Tetraethylendiamin
Tm Primer-Schmelztemperatur (Schmelztemperatur) TPA 12-o-Tetradecanoylphorbol-13-Acetat Tris Tris-(Hydroxymethyl)-Aminomethan
U Unit(s) oder Uracil UTP Uridintriphosphat
V Volt
v/v Volumen pro Volumen w/v Gewicht (weight) pro Volumen
X-Gal 5-Brom-4-Chlor-3-indolyl-β-glucuronid
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